Mapping of the Hif1a exon I.2 transcription initiation sites. (A) Primer extension. Poly(A)⁺ mRNA was isolated from mouse Hepa1 cells and annealed to the endlabeled, complementary oligonucleotide mHIFpex2 (see Fig 3). The primer was extended with reverse transcriptase and the products were resolved on a 6% denaturing polyacrylamide gel together with a sequencing reaction performed with the same oligonucleotide as primer and a plasmid containing the sequence shown in Fig 3 as template. (B) Nuclease protection. Total RNA derived from Hepa1 cells was hybridized to an endlabeled single-stranded antisense probe prepared as described in Materials and Methods. The DNA-RNA hybrids were treated with the indicated amounts of mung bean nuclease and separated on a sequencing gel along with an unrelated sequencing ladder that served as length marker. Numbers indicate the lengths of reaction products including the mHIFpex2 primer.